Figure 6: Effects of a gain of function of spry2 and U0126 treatment.

(a–h) When the MAP kinase cascade was blocked by injection of a target protection (TP) MO of spry2 (b,f) or U0126-treatment (d,h), hearts (dotted outlines) exhibited incomplete looping (f,h). Red arrowhead indicates pooling of blood, a sign of stagnation of circulation. The normal looped heart is shown in (e). (i–w) As observed in the miR-21 morphants and the arrested hearts, the endocardial cells were single-layered, flattened and stretched in the hearts injected with the TP MO of spry2 (n–r) and the U0126-treated embryos (s–w). Normal thickening of cells observed in the WT (i–m) was lost, as visualized by fli1-GFP reporter and Dm-grasp staining. As illustrated in a schematic representation (m, r and w), the multi-layered cuboidal cells observed in the WT (m) was detected neither in the hearts injected with the target protection MO of spry2 (r) or treated by U0126 (w). Scale bars, 500 μm (a–d), 50 μm (i,j,n,o,s,t).